SITE-DIRECTED MUTAGENESIS OF NONMUSCLE MYOSIN HEAVY CHAINS

我们已经成功地表达了mg数量的 鸡脑非肌肉肌球蛋白重链。 这个片段 从氨基末端残基开始并延伸到肌球蛋白尾部， 根据十二烷基硫酸钠中的Mr判断，约为110，000 kDa， 聚丙烯酰胺凝胶电泳 我们表达了这个片段 脑肌球蛋白重链的杆状病毒表达系统。 的 肌球蛋白重链可以被看作是一个突出的带在提取物中， 病毒感染的军虫肠细胞，并通过 分子筛色谱法 它的身份得到了西方的证实。 使用已知识别表位的单克隆抗体进行印迹， 肌球蛋白分子的头部区域。 它被发现含有 约10%的预期ATP酶活性和这种差的活性 归因于新生肌球蛋白重链的错误折叠。 因此，我们目前正在工程化表达17 kDa肌球蛋白 使用杆状病毒表达系统，并计划 共表达重链和轻链以获得肌球蛋白 片段具有完整的，尽管不受调节的肌球蛋白ATP酶活性。 未来 实验将针对突变肌球蛋白的头部区域， 分子，以确定各种氨基酸残基， 有助于肌球蛋白的生物活性。

We have succeeded in expressing mg quantities of the head fragment of the chicken brain nonmuscle myosin heavy chain. This fragment which begins at the amino terminal residue and extends into the myosin tail is approximately 110,000 kDa as judged by its Mr in sodium dodecyl sulfate- polyacrylamide gel electrophoresis. We expressed this fragment of the brain myosin heavy chain using the baculovirus expression system. The myosin heavy chain could be seen as a prominent band in extracts of the virally infected army worm intestinal cells, and was purified by molecular sieve chromatography. Its identity was confirmed by Western blotting using a monoclonal antibody known to recognize an epitope in the head region of the myosin molecule. It was found to contain approximately 10% of the expected ATPase activity and this poor activity was attributed to misfolding of the nascent myosin heavy chain. Therefore, we are presently engineering expression of the 17 kDa myosin light chain using the baculovirus expression system and plan to coexpress both heavy and light chain in an effort to obtain a myosin fragment with full, albeit unregulated, myosin ATPase activity. Future experiments will be directed at mutating the head region of the myosin molecule in an effort to identify the various amino acid residues that contribute to myosin's biological activity.